Generation and Characterization of a genetic zebrafish model of SMA carrying the human SMN2 gene

<p>Abstract</p> <p>Background</p> <p>Animal models of human diseases are essential as they allow analysis of the disease process at the cellular level and can advance therapeutics by serving as a tool for drug screening and target validation. Here we report the development of a complete genetic model of spinal muscular atrophy (SMA) in the vertebrate zebrafish to complement existing zebrafish, mouse, and invertebrate models and show its utility for testing compounds that alter <it>SMN2 </it>splicing.</p> <p>Results</p> <p>The human motoneuron disease SMA is caused by low levels, as opposed to a complete absence, of the survival motor neuron protein (SMN). To generate a true model of SMA in zebrafish, we have generated a transgenic zebrafish expressing the human <it>SMN2 </it>gene (<it>hSMN2</it>), which produces only a low amount of full-length SMN, and crossed this onto the <it>smn</it>^-/-background. We show that human <it>SMN2 </it>is spliced in zebrafish as it is in humans and makes low levels of SMN protein. Moreover, we show that an antisense oligonucleotide that enhances correct <it>hSMN2 </it>splicing increases full-length <it>hSMN </it>RNA in this model. When we placed this transgene on the <it>smn </it>mutant background it rescued the neuromuscular presynaptic SV2 defect that occurs in <it>smn </it>mutants and increased their survival.</p> <p>Conclusions</p> <p>We have generated a transgenic fish carrying the human <it>hSMN2 </it>gene. This gene is spliced in fish as it is in humans and mice suggesting a conserved splicing mechanism in these vertebrates. Moreover, antisense targeting of an intronic splicing silencer site increased the amount of full length SMN generated from this transgene. Having this transgene on the <it>smn </it>mutant fish rescued the presynaptic defect and increased survival. This model of zebrafish SMA has all of the components of human SMA and can thus be used to understand motoneuron dysfunction in SMA, can be used as an vivo test for drugs or antisense approaches that increase full-length SMN, and can be developed for drug screening.</p

VEGFR2 Expression Correlates with Postnatal Development of Brain Arteriovenous Malformations in a Mouse Model of Type I Hereditary Hemorrhagic Telangiectasia

\ua9 2023 by the authors.Brain arteriovenous malformations (BAVMs) are a critical concern in hereditary hemorrhagic telangiectasia (HHT) patients, carrying the risk of life-threatening intracranial hemorrhage. While traditionally seen as congenital, the debate continues due to documented de novo cases. Our primary goal was to identify the precise postnatal window in which deletion of the HHT gene Endoglin (Eng) triggers BAVM development. We employed SclCreER(+);Eng2f/2f mice, enabling timed Eng gene deletion in endothelial cells via tamoxifen. Tamoxifen was given during four postnatal periods: P1–3, P8–10, P15–17, and P22–24. BAVM development was assessed at 2–3 months using latex dye perfusion. We examined the angiogenic activity by assessing vascular endothelial growth factor receptor 2 (VEGFR2) expression via Western blotting and Flk1-LacZ reporter mice. Longitudinal magnetic resonance angiography (MRA) was conducted up to 9 months. BAVMs emerged in 88% (P1–3), 86% (P8–10), and 55% (P15–17) of cases, with varying localization. Notably, the P22–24 group did not develop BAVMs but exhibited skin AVMs. VEGFR2 expression peaked in the initial 2 postnatal weeks, coinciding with BAVM onset. These findings support the “second hit” theory, highlighting the role of early postnatal angiogenesis in initiating BAVM development in HHT type I mice

Absence of gemin5 from SMN complexes in nuclear Cajal bodies

<p>Abstract</p> <p>Background</p> <p>Spinal muscular atrophy is caused by reduced levels of the survival of motor neurons (SMN) protein. SMN is found in large complexes with Sm proteins and at least eight other proteins, including seven "gemins". These complexes are involved in the assembly of snRNPs in the cytoplasm and their transport into the nucleus, but the precise roles of the individual protein components are largely unknown.</p> <p>Results</p> <p>We have investigated the subcellular distribution of gemins using novel antibodies against gemins 3–7, and existing mAbs against SMN, gemin2, unrip, fibrillarin and profilin II. Most gemins were equally distributed between nuclear and cytoplasmic fractions of HeLa cells, but gemin5 and unrip were more abundant in the cytoplasm. In a cytoplasmic extract obtained by mild disruption of HeLa cells, nearly all the SMN and gemins 2–4 were in large complexes, but most of the gemin5 sedimented separately with a lower S value. Most of the unrip sedimented with gemins 6 and 7 near the top of the sucrose density gradients, separate from both SMN and gemin5. Anti-SMN mAbs pulled down gemin5 from cytoplasmic extracts, but not from nuclear extracts, and gemin5 did not co-sediment with large SMN complexes in nuclear extracts. These data suggest that gemin5 is easily detached from SMN-gemin complexes in the nucleus. By immuno-histochemistry, gemin5 was rarely detectable in nuclear gems/Cajal bodies, although it was accessible to antibody and easily detectable when present. This suggests that gemin5 is normally absent from SMN complexes in these nuclear storage sites.</p> <p>Conclusion</p> <p>We conclude that SMN complexes usually exist without gemin5 in nuclear gems/Cajal bodies. Gemin5 is believed to be involved in capturing snRNA into SMN complexes in the cytoplasm for transport into the nucleus. We hypothesize that gemin5, though present in the nucleus, is no longer needed for SMN complex function during the time these complexes are stored in gems/Cajal bodies.</p

Endothelial Depletion of Acvrl1 in Mice Leads to Arteriovenous Malformations Associated with Reduced Endoglin Expression

Rare inherited cardiovascular diseases are frequently caused by mutations in genes that are essential for the formation and/ or function of the cardiovasculature. Hereditary Haemorrhagic Telangiectasia is a familial disease of this type. The majority of patients carry mutations in either Endoglin (ENG) or ACVRL1 (also known as ALK1) genes, and the disease is characterized by arteriovenous malformations and persistent haemorrhage. ENG and ACVRL1 encode receptors for the TGF beta superfamily of ligands, that are essential for angiogenesis in early development but their roles are not fully understood. Our goal was to examine the role of Acvrl1 in vascular endothelial cells during vascular development and to determine whether loss of endothelial Acvrl1 leads to arteriovenous malformations. Acvrl1 was depleted in endothelial cells either in early postnatal life or in adult mice. Using the neonatal retinal plexus to examine angiogenesis, we observed that loss of endothelial Acvrl1 led to venous enlargement, vascular hyperbranching and arteriovenous malformations. These phenotypes were associated with loss of arterial Jag1 expression, decreased pSmad1/5/8 activity and increased endothelial cell proliferation. We found that Endoglin was markedly down-regulated in Acvrl1-depleted ECs showing endoglin expression to be downstream of Acvrl1 signalling in vivo. Endothelial-specific depletion of Acvrl1 in pups also led to pulmonary haemorrhage, but in adult mice resulted in caecal haemorrhage and fatal anaemia. We conclude that during development, endothelial Acvrl1 plays an essential role to regulate endothelial cell proliferation and arterial identity during angiogenesis, whilst in adult life endothelial Acvrl1 is required to maintain vascular integrity

Exogenous Transforming Growth Factor-β1 and Its Helminth-Derived Mimic Attenuate the Heart\u27s Inflammatory Response to Ischemic Injury and Reduce Mature Scar Size

\ua9 2024 American Society for Investigative Pathology. Coronary reperfusion after acute ST-elevation myocardial infarction (STEMI) is standard therapy to salvage ischemic heart muscle. However, subsequent inflammatory responses within the infarct lead to further loss of viable myocardium. Transforming growth factor (TGF)-β1 is a potent anti-inflammatory cytokine released in response to tissue injury. The aim of this study was to investigate the protective effects of TGF-β1 after MI. In patients with STEMI, there was a significant correlation (P = 0.003) between higher circulating TGF-β1 levels at 24 hours after MI and a reduction in infarct size after 3 months, suggesting a protective role of early increase in circulating TGF-β1. A mouse model of cardiac ischemia reperfusion was used to demonstrate multiple benefits of exogenous TGF-β1 delivered in the acute phase. It led to a significantly smaller infarct size (30% reduction, P = 0.025), reduced inflammatory infiltrate (28% reduction, P = 0.015), lower intracardiac expression of inflammatory cytokines IL-1β and chemokine (C-C motif) ligand 2 (&gt;50% reduction, P = 0.038 and 0.0004, respectively) at 24 hours, and reduced scar size at 4 weeks (21% reduction, P = 0.015) after reperfusion. Furthermore, a low-fibrogenic mimic of TGF-β1, secreted by the helminth parasite Heligmosomoides polygyrus, had an almost identical protective effect on injured mouse hearts. Finally, genetic studies indicated that this benefit was mediated by TGF-β signaling in the vascular endothelium

Identification of novel DNA methylation inhibitors via a two-component reporter gene system

<p>Abstract</p> <p>Background</p> <p>Targeting abnormal DNA methylation represents a therapeutically relevant strategy for cancer treatment as demonstrated by the US Food and Drug Administration approval of the DNA methyltransferase inhibitors azacytidine and 5-aza-2'-deoxycytidine for the treatment of myelodysplastic syndromes. But their use is associated with increased incidences of bone marrow suppression. Alternatively, procainamide has emerged as a potential DNA demethylating agent for clinical translation. While procainamide is much safer than 5-aza-2'-deoxycytidine, it requires high concentrations to be effective in DNA demethylation in suppressing cancer cell growth. Thus, our laboratories have embarked on the pharmacological exploitation of procainamide to develop potent DNA methylation inhibitors through lead optimization.</p> <p>Methods</p> <p>We report the use of a DNA methylation two-component enhanced green fluorescent protein reporter system as a screening platform to identify novel DNA methylation inhibitors from a compound library containing procainamide derivatives.</p> <p>Results</p> <p>A lead agent IM25, which exhibits substantially higher potency in <it>GSTp1 </it>DNA demethylation with lower cytotoxicity in MCF7 cells relative to procainamide and 5-aza-2'-deoxycytidine, was identified by the screening platform.</p> <p>Conclusions</p> <p>Our data provide a proof-of-concept that procainamide could be pharmacologically exploited to develop novel DNA methylation inhibitors, of which the translational potential in cancer therapy/prevention is currently under investigation.</p

"Flogging dead horses": evaluating when have clinical trials achieved sufficiency and stability? A case study in cardiac rehabilitation

<p>Abstract</p> <p>Background</p> <p>Most systematic reviews conclude that another clinical trial is needed. Measures of sufficiency and stability may indicate whether this is true.</p> <p>Objectives: To show how evidence accumulated on centre-based versus home-based cardiac rehabilitation, including estimates of sufficiency and stability</p> <p>Methods</p> <p>Systematic reviews of clinical trials of home versus centre-based cardiac rehabilitation were used to develop a cumulative meta-analysis over time. We calculated the standardised mean difference (SMD) in effect, confidence intervals and indicators of sufficiency and stability. Sufficiency refers to whether the meta-analytic database adequately demonstrates that an intervention works - is statistically superior to another. It does this by assessing the number of studies with null results that would be required to make the meta-analytic effect non-statistically significant. Stability refers to whether the direction and size of the effect is stable as new studies are added to the meta-analysis.</p> <p>Results</p> <p>The standardised mean effect difference reduced over fourteen comparisons from a non-significant difference favouring home-based cardiac rehabilitation to a very small difference favouring hospital (SMD -0.10, 95% CI -0.32 to 0.13). This difference did not reach the sufficiency threshold (failsafe ratio 0.039 < 1) but did achieve the criteria for stability (cumulative slope 0.003 < 0.005).</p> <p>Conclusions</p> <p>The evidence points to a relatively small effect difference which was stable but not sufficient in terms of the suggested thresholds. Sufficiency should arguably be based on substantive significance and decided by patients. Research on patient preferences should be the priority. Sufficiency and stability measures are useful tools that need to be tested in further case studies.</p

Analysing public service outsourcing: the value of a regulatory perspective

This article draws on findings from two longitudinal case studies of voluntary organisations engaged in delivering social care services via purchaser – provider relations with local authorities. The study focuses on changes to contractual relations, employment conditions in provider organisations and service quality. The article argues the influence of the market on these changes can only be adequately comprehended by rooting the analysis in an understanding of the way in which surrounding regulatory frameworks shape its structure and operation. In doing so, it reveals how in an era of shifting market conditions characterised by greater competition and dramatic local authority cuts, a ‘soft’ regulatory framework offers little support to partnership relations between voluntary organisations and local authorities. Instead, the regulatory environment undermines financial security among voluntary organisations, degrades employment conditions in them and raises concerns regarding their service quality